Identification of an essential spinoparabrachial pathway for mechanical itch.

The sensation of itch is a protective response that is elicited by either mechanical or chemical stimuli. The neural pathways for itch transmission in the skin and spinal cord have been characterized previously, but the ascending pathways that transmit sensory information to the brain to evoke itch perception have not been identified. Here, we show that spinoparabrachial neurons co-expressing Calcrl and Lbx1 are essential for generating scratching responses to mechanical itch stimuli. Moreover, we find that mechanical and chemical itch are transmitted by separate ascending pathways to the parabrachial nucleus, where they engage separate populations of FoxP2PBN neurons to drive scratching behavior. In addition to revealing the architecture of the itch transmission circuitry required for protective scratching in healthy animals, we identify the cellular mechanisms underlying pathological itch by showing the ascending pathways for mechanical and chemical itch function cooperatively with the FoxP2PBN neurons to drive chronic itch and hyperknesis/alloknesis.

Spinal Neuropeptide Y1 Receptor-Expressing Neurons Form an Essential Excitatory Pathway for Mechanical Itch.

Acute itch can be generated by either chemical or mechanical stimuli, which activate separate pathways in the periphery and spinal cord. While substantial progress has been made in mapping the transmission pathway for chemical itch, the central pathway for mechanical itch remains obscure. Using complementary genetic and pharmacological manipulations, we show that excitatory neurons marked by the expression of the neuropeptide Y1 receptor (Y1Cre neurons) form an essential pathway in the dorsal spinal cord for the transmission of mechanical but not chemical itch. Ablating or silencing the Y1Cre neurons abrogates mechanical itch, while chemogenetic activation induces scratching. Moreover, using Y1 conditional knockout mice, we demonstrate that endogenous neuropeptide Y (NPY) acts via dorsal-horn Y1-expressing neurons to suppress light punctate touch and mechanical itch stimuli. NPY-Y1 signaling thus regulates the transmission of innocuous tactile information by establishing biologically relevant thresholds for touch discrimination and mechanical itch reflexes.

Modulation of spinal motor networks by astrocyte-derived adenosine is dependent on D1-like dopamine receptor signaling.

Astrocytes modulate many neuronal networks, including spinal networks responsible for the generation of locomotor behavior. Astrocytic modulation of spinal motor circuits involves release of ATP from astrocytes, hydrolysis of ATP to adenosine, and subsequent activation of neuronal A1 adenosine receptors (A1Rs). The net effect of this pathway is a reduction in the frequency of locomotor-related activity. Recently, it was proposed that A1Rs modulate burst frequency by blocking the D1-like dopamine receptor (D1LR) signaling pathway; however, adenosine also modulates ventral horn circuits by dopamine-independent pathways. Here, we demonstrate that adenosine produced upon astrocytic stimulation modulates locomotor-related activity by counteracting the excitatory effects of D1LR signaling and does not act by previously described dopamine-independent pathways. In spinal cord preparations from postnatal mice, a D1LR agonist, SKF 38393, increased the frequency of locomotor-related bursting induced by 5-hydroxytryptamine and N-methyl-d-aspartate. Bath-applied adenosine reduced burst frequency only in the presence of SKF 38393, as did adenosine produced after activation of protease-activated receptor-1 to stimulate astrocytes. Furthermore, the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine enhanced burst frequency only in the presence of SKF 38393, indicating that endogenous adenosine produced by astrocytes during network activity also acts by modulating D1LR signaling. Finally, modulation of bursting by adenosine released upon stimulation of astrocytes was blocked by protein kinase inhibitor-(14-22) amide, a protein kinase A (PKA) inhibitor, consistent with A1R-mediated antagonism of the D1LR/adenylyl cyclase/PKA pathway. Together, these findings support a novel, astrocytic mechanism of metamodulation within the mammalian spinal cord, highlighting the complexity of the molecular interactions that specify motor output. NEW & NOTEWORTHY Astrocytes within the spinal cord produce adenosine during ongoing locomotor-related activity or when experimentally stimulated. Here, we show that adenosine derived from astrocytes acts at A1 receptors to inhibit a pathway by which D1-like receptors enhance the frequency of locomotor-related bursting. These data support a novel form of metamodulation within the mammalian spinal cord, enhancing our understanding of neuron-astrocyte interactions and their importance in shaping network activity.

Spinal Circuits for Touch, Pain, and Itch.

The exteroceptive somatosensory system is important for reflexive and adaptive behaviors and for the dynamic control of movement in response to external stimuli. This review outlines recent efforts using genetic approaches in the mouse to map the spinal cord circuits that transmit and gate the cutaneous somatosensory modalities of touch, pain, and itch. Recent studies have revealed an underlying modular architecture in which nociceptive, pruritic, and innocuous stimuli are processed by distinct molecularly defined interneuron cell types. These include excitatory populations that transmit information about both innocuous and painful touch and inhibitory populations that serve as a gate to prevent innocuous stimuli from activating the nociceptive and pruritic transmission pathways. By dissecting the cellular composition of dorsal-horn networks, studies are beginning to elucidate the intricate computational logic of somatosensory transformation in health and disease.

Gliotransmission and adenosinergic modulation: insights from mammalian spinal motor networks.

Astrocytes are proposed to converse with neurons at tripartite synapses, detecting neurotransmitter release and responding with release of gliotransmitters, which in turn modulate synaptic strength and neuronal excitability. However, a paucity of evidence from behavioral studies calls into question the importance of gliotransmission for the operation of the nervous system in healthy animals. Central pattern generator (CPG) networks in the spinal cord and brain stem coordinate the activation of muscles during stereotyped activities such as locomotion, inspiration, and mastication and may therefore provide tractable models in which to assess the contribution of gliotransmission to behaviorally relevant neural activity. We review evidence for gliotransmission within spinal locomotor networks, including studies indicating that adenosine derived from astrocytes regulates the speed of locomotor activity via metamodulation of dopamine signaling.

Differential regulation of NMDA receptors by d-serine and glycine in mammalian spinal locomotor networks.

Activation of N-methyl-d-aspartate receptors (NMDARs) requires the binding of a coagonist, either d-serine or glycine, in addition to glutamate. Changes in occupancy of the coagonist binding site are proposed to modulate neural networks including those controlling swimming in frog tadpoles. Here, we characterize regulation of the NMDAR coagonist binding site in mammalian spinal locomotor networks. Blockade of NMDARs by d(-)-2-amino-5-phosphonopentanoic acid (d-APV) or 5,7-dichlorokynurenic acid reduced the frequency and amplitude of pharmacologically induced locomotor-related activity recorded from the ventral roots of spinal-cord preparations from neonatal mice. Furthermore, d-APV abolished synchronous activity induced by blockade of inhibitory transmission. These results demonstrate an important role for NMDARs in murine locomotor networks. Bath-applied d-serine enhanced the frequency of locomotor-related but not disinhibited bursting, indicating that coagonist binding sites are saturated during the latter but not the former mode of activity. Depletion of endogenous d-serine by d-amino acid oxidase or the serine-racemase inhibitor erythro-ß-hydroxy-l-aspartic acid (HOAsp) increased the frequency of locomotor-related activity, whereas application of l-serine to enhance endogenous d-serine synthesis reduced burst frequency, suggesting a requirement for d-serine at a subset of synapses onto inhibitory interneurons. Consistent with this, HOAsp was ineffective during disinhibited activity. Bath-applied glycine (1-100 µM) failed to alter locomotor-related activity, whereas ALX 5407, a selective inhibitor of glycine transporter-1 (GlyT1), enhanced burst frequency, supporting a role for GlyT1 in NMDAR regulation. Together these findings indicate activity-dependent and synapse-specific regulation of the coagonist binding site within spinal locomotor networks, illustrating the importance of NMDAR regulation in shaping motor output.NEW & NOTEWORTHY We provide evidence that NMDARs within murine spinal locomotor networks determine the frequency and amplitude of ongoing locomotor-related activity in vitro and that NMDARs are regulated by d-serine and glycine in a synapse-specific and activity-dependent manner. In addition, glycine transporter-1 is shown to be an important regulator of NMDARs during locomotor-related activity. These results show how excitatory transmission can be tuned to diversify the output repertoire of spinal locomotor networks in mammals.

Stimulation of Glia Reveals Modulation of Mammalian Spinal Motor Networks by Adenosine.

Despite considerable evidence that glia can release modulators to influence the excitability of neighbouring neurons, the importance of gliotransmission for the operation of neural networks and in shaping behaviour remains controversial. Here we characterise the contribution of glia to the modulation of the mammalian spinal central pattern generator for locomotion, the output of which is directly relatable to a defined behaviour. Glia were stimulated by specific activation of protease-activated receptor-1 (PAR1), an endogenous G-protein coupled receptor preferentially expressed by spinal glia during ongoing activity of the spinal central pattern generator for locomotion. Selective activation of PAR1 by the agonist TFLLR resulted in a reversible reduction in the frequency of locomotor-related bursting recorded from ventral roots of spinal cord preparations isolated from neonatal mice. In the presence of the gliotoxins methionine sulfoximine or fluoroacetate, TFLLR had no effect, confirming the specificity of PAR1 activation to glia. The modulation of burst frequency upon PAR1 activation was blocked by the non-selective adenosine-receptor antagonist theophylline and by the A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, but not by the A2A-receptor antagonist SCH5826, indicating production of extracellular adenosine upon glial stimulation, followed by A1-receptor mediated inhibition of neuronal activity. Modulation of network output following glial stimulation was also blocked by the ectonucleotidase inhibitor ARL67156, indicating glial release of ATP and its subsequent degradation to adenosine rather than direct release of adenosine. Glial stimulation had no effect on rhythmic activity recorded following blockade of inhibitory transmission, suggesting that glial cell-derived adenosine acts via inhibitory circuit components to modulate locomotor-related output. Finally, the modulation of network output by endogenous adenosine was found to scale with the frequency of network activity, implying activity-dependent release of adenosine. Together, these data indicate that glia play an active role in the modulation of mammalian locomotor networks, providing negative feedback control that may stabilise network activity.

Discovery of AZD3514, a small-molecule androgen receptor downregulator for treatment of advanced prostate cancer.

Removal of the basic piperazine nitrogen atom, introduction of a solubilising end group and partial reduction of the triazolopyridazine moiety in the previously-described lead androgen receptor downregulator 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (1) addressed hERG and physical property issues, and led to clinical candidate 6-(4-{4-[2-(4-acetylpiperazin-1-yl)ethoxy]phenyl}piperidin-1-yl)-3-(trifluoromethyl)-7,8-dihydro[1,2,4]triazolo[4,3-b]pyridazine (12), designated AZD3514, that is being evaluated in a Phase I clinical trial in patients with castrate-resistant prostate cancer.

Small-molecule androgen receptor downregulators as an approach to treatment of advanced prostate cancer.

Chemical starting points were investigated for downregulation of the androgen receptor as an approach to treatment of advanced prostate cancer. Although prototypic steroidal downregulators such as 6a designed for intramuscular administration showed insufficient cellular potency, a medicinal chemistry program derived from a novel androgen receptor ligand 8a led to 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (10b), for which high plasma levels following oral administration in a preclinical model compensate for moderate cellular potency.

Bilateral Killian-Jamieson diverticula: a case report and literature review.

A Killian-Jamieson diverticulum is an outpouching from the lateral wall of the proximal cervical esophagus. These diverticula are rare and are distinct from the more commonly known Zenker's diverticulum. Literature regarding Killian-Jamieson diverticula and its suggested management is scarce. The present report describes a patient with symptomatic bilateral Killian-Jamieson diverticula. The patient had both diverticula excised and an esophagomyotomy performed. Following surgery, the patient's symptoms resolved and he recovered well. A literature review and discussion of the etiology, clinical presentation and radiographic findings of Killian-Jamieson diverticulum follow, as do recommendations for clinical management.

Imidazole piperazines: SAR and development of a potent class of cyclin-dependent kinase inhibitors with a novel binding mode.

A piperazine series of cyclin-dependent kinase (CDK) inhibitors have been identified. The compounds exhibit excellent physiochemical properties and a novel binding mode, whereby a bridging interaction via a water molecule with Asp 86 of CDK2, leads to selectivity for the CDK family of enzymes over other kinases. Piperazines 2e and 2i were subsequently shown to inhibit tumour growth when dosed orally in a nude mouse xenograft study. Additional chemical series that exploit this unexpected interaction with Asp 86 are also described.

Neutral 5-substituted 4-indazolylaminoquinazolines as potent, orally active inhibitors of erbB2 receptor tyrosine kinase.

We have identified a new series of C-5 substituted indazolylaminoquinazolines as potent erbB2 kinase inhibitors. The lead compound 22 showed excellent in vitro potency, good physical properties, acceptable oral pharmacokinetics in rat and dog, and low human in vitro clearance. It showed at least equivalent activity dose for dose compared to lapatinib in various erbB2- or EGFR-driven xenograft models after chronic oral administration.

New classes of antibacterial oxazolidinones with C-5, methylene O-linked heterocyclic side chains.

Exploration of the structure-activity relationships of the traditional C-5 acetamidomethyl side chain of the oxazolidonone antibacterials has yielded new, potent series of compounds of which the first examples, the O-linked iosoxazoles are described in detail, leading to the selection of the pre-clinical candidate AZD2563.